Prevascularized constructs for implantation to provide blood perfusion

ABSTRACT

This application discloses methods and materials for preparing functional microvascular beds in the laboratory. These prevascularized constructs can be used to vascularize engineered tissue constructs or to revascularize damaged or diseased tissues or organs following implantation. The prevascularized constructs may also deliver genetically engineered gene products to the bloodstream.

RELATED APPLICATION INFORMATION

This application claims the filing date benefit from, and is adivisional application of, U.S. patent application Ser. No. 10/112,461(now U.S. Pat. No. 7,029,838), which claimed priority to U.S.Provisional Patent Application Ser. No. 60/279,824, filed Mar. 30, 2001.

DESCRIPTION OF THE INVENTION

Field of the Invention

The invention is directed generally to methods for vascularizing orrevascularizing tissues and organs, including but not limited to donatedtissues and organs, diseased or dysfunctional tissues and organs, andengineered tissues. Prevascularized constructs comprising microvesselfragments in a three dimensional culture, for use according to thesemethods, are also disclosed. Prevascularized constructs comprisinggenetically engineered cells for delivering a gene(s) or gene product(s)to an animal or human are also provided.

Background of the Invention

Transplants of cells and tissue engineered organs and tissues offerpromise in facilitating tissue healing and repair and the replacement ortreatment of diseased or dysfunctional organs. A primary challenge inthe transplantation of tissue engineered constructs is ensuringsufficient blood supply to the constituent cells. In the absence ofpre-existing vessels in the transplant capable of inosculation with therecipient blood supply, the amount of tissue that can be transplanted islimited by oxygen diffusion.

Ultimately, healthy transplants depend on sufficient vessel densitywithin the transplanted tissue or organ and the organization of thevessels into a network comprised of low-resistance conduit vessels(arteries), a functional microcirculation (arterioles and capillaries)for a proper blood-tissue exchange, and drainage/compliance vessels(venules and veins). Thus, in certain respects, the extent to whichtissue engineered constructs can be vascularized determines the limitsof construct size and architecture. Consequently, techniques to providerapid vascularization and perfusion of tissue engineered constructsshould be incorporated into the tissue or organ design.

The ability to include a vascular network within an engineered tissueand engineer it to match a particular tissue represents a significantstride in the tissue engineering and artificial organ fields. A recentarticle in the “Red Herring”, a leading technology layman's journal,described the pre-vascularization of an engineered organ to be theremaining, but most difficult, step in successfully growing organs inthe laboratory.

Current tissue engineering strategies are hampered by the inability topre-build a vasculature into the tissue. Any implanted tissue,engineered or donated, requires a blood supply in order to support thehealth and function of the tissue cells. In the case of most built orengineered tissues (tissue engineering), there is no vasculature withinthe construct to perform this function.

Existing strategies for building a vascular system for tissue engineeredconstructs have been based on using cultured, human endothelial cells.Researchers were successful in generating a functional vascular tree,but only in a complex, ill-defined experimental gel scaffold. It wasnecessary to add a number of differentiation-promoting factors, some ofwhich are not well defined, in order to induce the isolated single cellsinto a vessel structure (Lawley T J and Kubato Y J. Invest Derm.93:59S-61S 1989, Schechner J S et al. PNAS 97:9191-9196, 2000). In onestudy, it was necessary to transfect a gene that blocked apoptosis (orprogrammed cell death) into cultured endothelial cells in order for thevasculature to persist in the implant (Nor J E et al., Lab Invest.81:453-463 2001; Schechner J S et al. PNAS 97:9191-9196, 2000).

Additionally, in tissues and organs suffering from the consequences ofchronic ischemic disease such as after myocardial infarction orperipheral vascular disease, expansion of the vasculature adjacent tothe effected tissue areas into the ischemic zones offers one mechanismby which these tissues can be recovered. Thus, there exists a need formethods and compositions for vascularizing tissue engineered constructsand donated tissues and organs and for revascularizing diseased ordysfunctional tissues and organs. Preferably, such methods and materialswould not require the incorporation of genetically engineered cells toavoid premature apoptosis.

SUMMARY OF THE INVENTION

This application discloses methods and materials by which functionalmicrovascular beds can be preformed in the laboratory for use inengineered tissue constructs and therapeutic applications. According tocertain embodiments of the invention, a fully developed vascular networkforms and persists in a simple, host-derived matrix and without the needto genetically engineer the vessels. The core technology of the processinvolves the expansion of a collection of isolated capillary fragmentsinto an intact, functional capillary bed. Capillary fragments arepurified and collected from a vascular tissue, usually adipose tissue,and placed into a three dimensional culture environment that, in certainembodiments, comprises a collagen I gel (Hoying et al., In Vitro32:409-419, 1996). These fragments sprout and grow by day 4 in thisculture environment, ultimately establishing a new microvascular(capillary) bed by day 11 (see FIG. 1). This new vasculature has all ofthe structural and cellular features of a viable capillary bed observedin the body (Id.). Cultured vessels are on average 15 microns in outerdiameter, contain patent lumen, have endothelial cells oriented alongthe long axis of the vessels and extra-capillary mural cells coveringthe endothelial cell-formed tubes.

In certain embodiments, the present invention provides engineeredmicrovascular networks, also referred to as “prevascularizedconstructs”, that will connect with the vasculature of a host animalfollowing implantation and carry blood (see FIG. 2). Furthermore, thecapillaries present in the construct prior to implantation subsequentlyresult in the formation of a mature and functional vascular network(arteries, arterioles, capillaries and veins) required for proper tissueperfusion and health (see FIG. 3). This is supported by data obtained inexperimental animal models.

The new capillary beds can be generated from rat-derived vesselfragments. However human-derived vessel fragments may also be used inthe same process. Additionally, the inventors envision a process bywhich microvessel fragments are isolated from a patient's adipose tissueharvested by liposuction. To isolated microvessel fragments, the methodof isolating single endothelial cells may be used as described in U.S.Pat. No. 5,957,972, the entire contents of which are incorporated byreference. Harvested vessel fragments would be placed into athree-dimensional culture using fibrin derived from the patient's ownblood as the 3-D matrix scaffold. In this manner, a patient wouldreceive his own (autologous) vessels after a brief culture period (e.g.,one hour to 30 days).

The methods and compositions disclosed herein may be used in tissueengineering, as an implant for stimulating angiogenesis in neighboringhost tissues, and as a means for delivering recombinant gene productsthroughout the body. In certain embodiments, these methods andcompositions include mixing in stromal cells with the vascular elementsand determining cell viability and function. Initial cell types to beexamined are muscle precursor stem cells and pancreas ∃-islet cells, toname a few. Furthermore, the nature of the resulting vasculature presentin the construct following implantation into a host animal can becharacterized. Additionally, a variety of scaffold matrices includingfibrin gels and artificial, FDA-approved synthetic biocompatiblepolymers may be used.

I. Prevascularization of Engineered Tissues and Organs.

In certain embodiments, the above-described process could beincorporated into tissue building methods to establish, prior toimplantation, a functional vasculature within a tissue engineered organor tissue. Characterization of the capillary bed formed in culture andthe resulting vasculature present after implantation indicates that thecultured vessels have the potential to differentiate or change into thetype of vasculature required to meet specific tissue needs. What thisimplies is that it may be possible to affect changes to this basic“foundation” microvasculature built in the lab and impart a newcharacter to the microvascular bed to match the type of tissue beingbuilt. For example, engineered heart muscle will have a relatively highcapillary density while the vasculature of a liver organoid will exhibita typical sinusoid-like character. The pre-vascularization processdisclosed herein has great potential to incorporate a vascular networkwithin an engineered tissue and engineer it to match a particular tissueof interest, thus overcoming a significant hurdle in tissue engineering.

Thus, methods for stimulating or inducing the vascularization ofengineered tissues are provided. In certain embodiments, theprevascularized construct is incorporated into the tissue engineeringprocess to create a functional vasculature within the engineered tissue.In other embodiments, at least one prevascular construct is placed on atleast one surface of, or adjacent to, at least one engineered tissue. Incertain embodiments, the engineered tissue(s) is vascularizedpost-implantation.

II. Stimulus for Tissue Revascularization.

In tissues suffering from the consequences of chronic ischemic disease,such as after myocardial infarction or peripheral vascular disease,expansion of the vasculature adjacent to the effected tissue areas intothe ischemic zones offers one mechanism by which these tissues can berecovered. Implantation of the prevascularized construct could act as astimulus and nidus for revascularization of the affected areas. In thisregard, the implant would act as a nucleus of vascular growth, rapidlyestablishing a new vascular network within the previously avascular or“hypo-vascular” zone. We have evidence that the presence of theengineered vessels preserves the surrounding tissue integrity (see FIG.3). We believe that insertion of these prevascularized constructs willnot only provide for a rapid reperfusion of injured tissues, but mayalso support the restructuring and repair of those tissues. Byincorporating stem cells, progenitor cells or Relevant Cells into theprevascularized construct, cells useful for restructuring, repairingand/or repopulating damaged tissues or organs are provided. In certainembodiments, methods for stimulating or inducing the revascularizationof at least one tissue or at least one organ are provided. In certainembodiments, the tissue or organ may be ischemic and/or have a zone orregion that is avascular or hypovascular, for example, but not limitedto, chronic ischemic disease such as after myocardial infarction,peripheral vascular disease, or cerbrovascular accident (stroke).

III. Gene Product Delivery.

Current gene therapy strategies suffer from difficulties in successfullygetting the desired gene incorporated into cells of the patient and thetherapeutic protein (produced by the recombinant gene) distributedthroughout the body. Use of these prevascularized constructs in genedelivery provides 1) a means by which genetically engineered cellsincluded in the tissue construct have ready access to a blood stream(molecular exchange to and from the blood stream occurs best incapillaries) and 2) the culture vessel elements themselves are amenableto genetic engineering and may act as the source of therapeutic geneproduct. Prevascularized constructs provide a potential means to solvingthis problem.

In certain embodiments, prevascularized constructs comprisinggenetically engineered cells are disclosed. Such prevascularizedconstructs comprising genetically engineered cells are useful in thevascularization and revascularization methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the expansion of microvessel fragments into a functionalcapillary bed. Vessel fragments undergo sprouting (day 4), as shown inthe upper right panel, and eventually grow into an elaborate capillarytree (day 11), as shown in the lower panel.

FIG. 2 depicts a prevascularized construct 14 days after implantation.The engineered tissue is flushed, indicating the presence of bloodwithin the simple tissue. Furthermore, a feed vessel has grown into theconstruct.

FIG. 3 shows the development of mature vessel elements into a functionalvasculature within the implant. The vasculature consists of inputvessels (artery), as indicated by the “artery” arrow in the right-handpanel, exchange vessels (capillary), and draining vessels (vein), asindicated by the “capillary” and “vein” arrows in the left-hand panel.

FIG. 4. Vessel fragments exhibit features of angiogenesis when culturedin 3-D collagen gels. A A freshly isolated arteriole fragment with acapillary branch suspended in the collagen gel (day 0). B,C The samevessel fragment shown in A at day 4 (B) and day 5 (C) of culturing.Multiple sprouts are present by day 4 some which may continue toelongate (arrow) or regress (*) by the next day. D A representativefield of neovessels (arrows) stained by immunofluorescence that haveformed within the collagen gel system by day 11 of culturing.

FIG. 5. Collagen gels containing cultured neovessels (prevascularizedconstruct) inosculate with the host vasculature and carry blood whenimplanted into scid/scid mice. A,B Gross views of a prevascularizedconstruct (A) and an avascular collagen gel (B) 7 days and 3 dayspost-implantation, respectively. The long arrow in A points to a largevessel entering the construct from the surrounding host tissue. C-FOrthogonal Polarized Spectroscopy (OPS) images of separateprevascularized implants at day 3 (C), day 7 (D), and day 14 (F) or aday 7, avascular control gel (E). OPS detects the presence ofhemoglobin.

FIG. 6. Histology of characteristic prevascularized constructs after 1day (A), 2 days (B) and 14 days (C-F) of subcutaneous implantation.(A-B) Constructs were perfusion-filled with ink to identify thosevessels within the construct connected to host vessels. (C-F) Red bloodcells are present within vessel-like compartments in all sections. Byday 14 capillaries, veins and arteries are present within the construct.

FIG. 7. Prevascularized implants contain vascular endothelium areexhibit little inflammation. A-B Sections of either day 5 (A) or day 28(B) implants stained for the rodent-specific endothelial cell marker,GS-1. C A section of day 5 implant stained for the presence ofinfiltrating macrophages (arrow). D Vessel density measurements countedfrom GS-1 stained sections of implanted prevascularized constructs.Vessel density increases in implanted constructs between day 5 and day28 post-implantation.

FIG. 8. Nearly all of the cells within the prevascularized constructsare derived from the original cultured isolates. Serial sections of day5 (B,D) and day 28 (A, C, E) implants were either stained withhematoxylin to identify all nuclei within the section or labeled by insitu hybridization using a probe for the Y chromosome to identify cellsfrom the original isolate (vessel fragments were isolated from male ratsand constructs implanted into female mice). A A low magnification of aday 28 implant labeled by in situ hybridization. The “m” depicts theunderlying host (mouse) musculature, while the “i” demarks the implant.F For each serial section pair, the number of Y chromosome-positivenuclei were compared to the total number of nuclei (counted from thehematoxylin-stained sections) and reported as % Y positive nuclei ineach section for day 5 and day 28 implants.

FIG. 9. Collagen gel implants that are vascularized by freshly isolatedhuman microvessel fragments are not replaced by host vasculature by day28 post-implantation. A A section of a human vascularized constructstained with the rodent specific, vascular marker GS-1. The lectinlabels vessels within the surrounding mouse tissue (arrows), but onlylimited numbers of vessels within the construct (large clear zone in thecenter of the section). In contrast, the human-specific lectin, UEA1labels only vessels (open and closed arrows) within the construct (A,low mag.; B, high mag.), but not within the surrounding host tissue.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All references cited in this application are expressly incorporated byreference for any purpose.

The term “three-dimensional culture” is used in the broad sense hereinand refers to a composition comprising a biocompatible matrix, scaffold,or the like. The three-dimensional culture may be liquid, gel,semi-solid, or solid at 25° C. The three-dimensional culture may bebiodegradable or non-biodegradable. Exemplary three-dimensional culturematerials include polymers and hydrogels comprising collagen, fibrin,chitosan, MATRIGEL, polyethylene glycol, dextrans including chemicallycrosslinkable or photocrosslinkable dextrans, and the like. In certainembodiments, the three-dimensional culture comprises allogeneiccomponents, autologous components, or both allogeneic components andautologous components. In certain embodiments, the three-dimensionalculture comprises synthetic or semi-synthetic materials. In certainembodiments, the three-dimensional culture comprises a framework orsupport, such as a fibrin-derived scaffold. The term “scaffold” is alsoused in a broad sense herein. Thus scaffolds include a wide variety ofthree-dimensional frameworks, for example, but not limited to a mesh,grid, sponge, foam, or the like.

The terms “engineered tissue”, “engineered tissue construct”, or “tissueengineered construct” as used herein refer to a tissue or organ that isproduced, in whole or in part, using tissue engineering techniques.Descriptions of these techniques can be found in, among other places,“Principles of Tissue Engineering, 2d ed.”, Lanza, Langer, and Vacanti,eds., Academic Press, 2000 (hereinafter “Lanza et al.”); “Methods ofTissue Engineering”, Atala and Lanza, eds., Academic Press, 2001(hereinafter “Atala et al.”); Animal Cell Culture, Masters, ed., OxfordUniversity Press, 2000, (hereinafter “Masters”), particularly Chapter 6;and U.S. Pat. No. 4,963,489 and related U.S. Patents.

The term “microvessel fragment” as used herein refers to a segment orpiece of vascular tissue, including at least a part or segment of atleast one artery, arteriole, capillary, venule, or vein. Typically amicrovessel includes endothelial cells arranged in a tube surrounded byone or more layers of mural cells, such as smooth muscle cells orpericytes, and may further comprise extracellular matrix components,such as basement membrane proteins. In certain embodiments, themicrovessel fragments are obtained from vascular tissue, for example,but not limited to, skin, skeletal muscle, cardiac muscle, the atrialappendage of the heart, lung, mesentery, or adipose tissue. In certainembodiments, the adipose tissue microvessel fragments are obtained from,for example, but not limited to, subcutaneous fat, perirenal fat,pericardial fat, omental fat, breast fat, epididymal fat, properitonealfat, and the like. The skilled artisan will appreciate that other fatdeposits or any vascular-rich tissue or organ may serve as a source ofmicrovessel fragments for use in the invention, for example, but notlimited to, skin, muscle, including skeletal or cardiac muscle, lung,and mesentery. In certain embodiments, the microvessel fragments areobtained from adipose tissue harvested by liposuction or abdominoplasty.Adipiose tissue harvested by a liposuction procedure where a sonic probeis not used during the harvesting process is particularly useful.

The terms “vascularize”, “vascularizing”, or “vascularization” as usedherein refer to providing a functional or substantially functionalvascular network to an organ or tissue, particularly an engineeredtissue. A functional or substantially functional vascular network is onethat perfuses or is capable of perfusing the tissue or organ to meetsome or all of the tissue's or organ's nutritional needs, oxygen demand,and waste product elimination needs. A vascular tissue is a naturaltissue that is rich in vascular elements, such as microvessels, forexample, but without limitation, adipose tissue.

The terms “revascularize”, “revascularizing”, “neovascularization”, or“revascularization” as used herein refer to revising an existingvascular network or establishing a new functional or substantiallyfunctional vascular network in a tissue or organ that has an avascularor hypovascular zone, typically due to disease, congenital defect, orinjury. Additionally, the topical application of certainchemotherapeutic agents, for example, but not limited to, 5-flourouracil(5-FU), may also result in an ischemic or avascular zone. Such anavascular or hypovascular tissue or organ is often totally or partiallydysfunctional or has limited function and may be in need ofrevascularization. Revascularizing such a tissue or organ may result inrestored or augmented function.

As used herein, the term “polymer” is used in the broad sense and isintended to include a wide range of biocompatible polymers, for example,but not limited to, homopolymers, co-polymers, block polymers,cross-linkable or crosslinked polymers, photoinitiated polymers,chemically initiated polymers, biodegradable polymers, nonbiodegradablepolymers, and the like. In other embodiments, the prevascularizedconstruct comprises a polymer matrix that is nonpolymerized, to allow itto be combined with a tissue, organ, or engineered tissue in a liquid orsemi-liquid state, for example, by injection. In certain embodiments,the prevascularized construct comprising liquid matrix may polymerize orsubstantially polymerize “in situ.” In certain embodiments, theprevascularized construct is polymerized or substantially polymerizedprior to injection. Such injectable compositions are prepared usingconventional materials and methods know in the art, including, but notlimited to, Knapp et al., Plastic and Reconstr. Surg. 60:389-405, 1977;Fagien, Plastic and Reconstr. Surg. 105: 362-73 and 2526-28, 2000; Kleinet al., J. Dermatol. Surg. Oncol. 10:519-22, 1984; Klein, J. Amer. Acad.Dermatol. 9:224-28, 1983; Watson et al., Cutis 31:543-46, 1983; Klein,Dermatol. Clin. 19:491-508, 2001; Klein, Pedriat. Dent. 21:449-50, 1999;Skorman, J. Foot Surg. 26:511-5, 1987; Burgess, Facial Plast. Surg.8:176-82, 1992; Laude et al., J. Biomech. Eng. 122:231-35, 2000; Frey etal., J. Urol. 154:812-15, 1995; Rosenblatt et al., Biomaterials15:985-95, 1994; Griffey et al., J. Biomed. Mater. Res. 58:10-15, 2001;Stenburg et al., Scfand. J. Urol. Nephrol. 33:355-61, 1999; Sclafani etal., Facial Plast. Surg. 16:29-34, 2000; Spira et al., Clin. Plast.Surg. 20:181-88, 1993; Ellis et al., Facila Plast. Surg. Clin. NorthAmer. 9:405-11, 2001; Alster et al., Plastic Reconstr. Surg.105:2515-28, 2000; and U.S. Pat. Nos. 3,949,073 and 5,709,854.

In certain embodiments, the polymerized or nonpolymerized matrixcomprises collagen, including contracted and non-contracted collagengels, hydrogels comprising, for example, but not limited to, fibrin,alginate, agarose, gelatin, hyaluronate, polyethylene glycol (PEG),dextrans, including dextrans that are suitable for chemicalcrosslinking, photocrosslinking, or both, albumin, polyacrylamide,polyglycolyic acid, polyvinyl chloride, polyvinyl alcohol,poly(n-vinyl-2-pyrollidone), poly(2-hydroxy ethyl methacrylate),hydrophilic polyurethanes, acrylic derivatives, pluronics, such aspolypropylene oxide and polyethylene oxide copolymer, or the like. Incertain embodiments, the fibrin or collagen is autologous or allogeneicwith respect to the intended recipient. The skilled artisan willappreciate that the matrix may comprise non-degradable materials, forexample, but not limited to, expanded polytetrafluoroethylene (ePTFE),polytetrafluoroethylene (PTFE), polyethyleneterephthalate (PET),polyurethane, polyethylene, polycabonate, polystyrene, silicone, and thelike, or selectively degradable materials, such as poly(lactic-co-glycolic acid; PLGA), PLA, or PGA. (See also, Middleton etal., Biomaterials 21:2335-2346, 2000; Middleton et al., Medical Plasticsand Biomaterials, March/April 1998, at pages 30-37; Handbook ofBiodegradable Polymers, Domb, Kost, and Domb, eds., 1997, HarwoodAcademic Publishers, Australia; Rogalla, Minim. Invasive Surg. Nurs.11:67-69, 1997; Klein, Facial Plast. Surg. Clin. North Amer. 9:205-18,2001; Klein et al., J. Dermatol. Surg. Oncol. 11:337-39, 1985; Frey etal., J. Urol. 154:812-15, 1995; Peters et al., J. Biomed. Mater. Res.43:422-27, 1998; and Kuijpers et al., J. Biomed. Mater. Res. 51:136-45,2000).

I. Prevascularized Constructs.

The terms “prevascularized construct” or “engineered microvascularnetwork” refers to a composition comprising at least one microvesselfragment, typically isolated from a vascular-rich tissue, in athree-dimensional culture, including but not limited to a matrix,scaffold, gel, or liquid. In certain embodiments, the prevascularizedconstructs comprise a three-dimensional matrix and microvesselfragments. In certain embodiments, the matrix comprises a preformedframework, for example, but not limited to a fibrin scaffold. In certainembodiments, the three-dimensional culture comprises a polymerized,substantially polymerized, or nonpolymerized matrix.

In certain embodiments, prevascularized constructs are prepared bycombining microvessel fragments and a liquid three-dimensional culture,such as nonpolymerized collagen, agarose, gelatin, other nonpolymerizedpolymer matrices, or the like. In other embodiments, the microvesselfragments are seeded, sodded or perfused onto or through a solid orsemi-solid three-dimensional culture environment, for example, but notlimited to, a framework, scaffold, hollow-fiber filter, or the like.

Prevascularized constructs may be categorized as “cultured microvesselconstructs” or “freshly isolated microvessel constructs.” A culturedmicrovessel construct is typically incubated prior to implantation. Forexample, but not limited to, in a humidified incubator at 37° C. and 5%CO₂. Typically such cultured microvessel constructs are incubated for aperiod of one hour to thirty days, but may be incubated for shorter orlonger periods, as desired. The skilled artisan will appreciate that theterm “cultured” may or may not refer to the use of conventionalincubation methods, such as a controlled-temperature incubator.Alternately, a prevascularized construct may comprise a freshly isolatedmicrovessel construct that undergoes little or no incubation prior touse. The skilled artisan will appreciate that freshly isolatedmicrovessel constructs may, but need not, be incubated. In certainembodiments, a freshly isolated microvessel construct comprisesmicrovessel fragments in a three-dimensional culture that has been“incubated” subsequent to the introduction of the microvessels, forexample, but without limitation, to allow the construct to polymerize.In other embodiments, a freshly isolated microvessel construct comprisesa liquid three-dimensional culture, as may be appropriate forimplantation by injection (see, e.g., U.S. Pat. Nos. 5,709,854 and6,224,893). Such liquid constructs may, but need not, polymerize in situunder appropriate conditions.

The skilled artisan will understand that prevascularized constructscomprising a nonpolymerized liquid three-dimensional culture that issubsequently allowed to polymerize or gel are capable of assuming amultitude of shapes. Thus, in certain embodiments, the ultimate size andshape of the polymerized construct depends, in part, on the size andshape of the vessel in which the construct is polymerized. For example,but not limited to, cylindrical or tubular constructs can be preparedusing conical tubes; disk-shaped constructs can be prepared usingmulti-well plates; planar constructs can be prepared using flatsurfaces, for example, a petri dish, the inverted lid of a multi-wellplate, or a flat-bottomed dish. Additionally, in certain embodiments,polymerized prevascularized constructs can be cut or trimmed into adesired size or shape. Thus, prevascularized constructs can be preparedin virtually any size and shape, prior to or during use.

In certain embodiments, the prevascularized construct comprisesautologous microvessel fragments in an autologous or substantiallyautologous three-dimensional culture. In certain embodiments,prevascularized constructs comprise microvessel fragments in athree-dimensional culture comprising a scaffold, for example, but notlimited to, fibrin-derived scaffolds (see, e.g., Nicosia et al., Lab.Invest. 63:115-22, 1990) and scaffolds comprising artificial,FDA-approved synthetic biocompatible polymers, for example, but notlimited to, polyethylene, polymethacrylate, polyurethane, vinyl, such aspolyvinyl chloride, silicones, PLGA, PTFE, ePTFE, polypropylene,polyethyleneterephthalate (PET), nylon, polylactide, and polyglycolide.Discussions of exemplary biocompatible polymers, scaffolds, and othermatrix materials, including protocols for their preparation and use, maybe found in, among other places, Atala et al., particularly Chapters42-76; Lanza et al., particularly Chapters 21 and 22; and Handbook ofBiodegradable Polymers, Domb, Kost, and Domb, eds., 1997, HarwoodAcademic Publishers, Australia.

In certain embodiments, the prevascularized constructs comprisemicrovessel fragments that are autologous or allogeneic with respect tothe intended human or animal recipient. In certain embodiments, theprevascularized construct further comprises at least one cytokine, atleast one chemokine, at least one antibiotic, such as an antimicrobialagent, at least one drug, at least one analgesic agent, at least oneanti-inflammatory agent, at least one immunosuppressive agent, orvarious combinations thereof. In certain embodiments, the at least onecytokine, at least one antibiotic, at least one drug, at least oneanalgesic agent, at least one anti-inflammatory agent, at least oneimmunosuppressive agent, or various combinations thereof comprise acontrolled-release format, such as those generally known in the art, forexample, but not limited to, Richardson et al., Nat. Biotechnol.19:1029-34, 2001.

Exemplary cytokines include angiogenin, vascular endothelial growthfactor (VEGF, including, but not limited to VEGF-165), interleukins,fibroblast growth factors, for example, but not limited to, FGF-1 andFGF-2, hepatocyte growth factor, (HGF), transforming growth factor beta(TGF-β), endothelins (such as ET-1, ET-2, and ET-3), insulin-like growthfactor (IGF-1), angiopoietins (such as Ang-1, Ang-2, Ang-3/4),angiopoietin-like proteins (such as ANGPTL1, ANGPTL-2, ANGPTL-3, andANGPTL-4), platelet-derived growth factor (PDGF), including, but notlimited to PDGF-M, PDGF-BB and PDGF-AB, epidermal growth factor (EGF),endothelial cell growth factor (ECGF), including ECGS, platelet-derivedendothelial cell growth factor (PD-ECGF), placenta growth factor (PLGF),and the like. Cytokines, including recombinant cytokines, and chemokinesare typically commercially available from numerous sources, for example,R & D Systems (Minneapolis, Minn.); Endogen (Woburn, Wash.); and Sigma(St. Louis, Mo.). The skilled artisan will understand that the choice ofchemokines and cytokines for incorporation into particularprevascularized constructs will depend, in part, on the target tissue ororgan to be vascularized or revascularized.

In certain embodiments, prevascularized constructs further comprise atleast one genetically engineered cell. In certain embodiments,prevascularized constructs comprising at least one geneticallyengineered cell will constitutively express or inducibly express atleast one gene product encoded by the at least one geneticallyengineered cell due to the genetic alterations within the at least onegenetically engineered cell induced by techniques known in the art.Descriptions of exemplary genetic engineering techniques can be foundin, among other places, Ausubel et al., Current Protocols in MolecularBiology (including supplements through March 2002), John Wiley & Sons,New York, N.Y., 1989; Sambrook et al., Molecular Cloning: A LaboratoryManual, 2^(nd) Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989; Sambrook and Russell, Molecular Cloning: ALaboratory Manual, 3^(rd) Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001; Beaucage et al., Current Protocols in NucleicAcid Chemistry, John Wiley & Sons, New York, N.Y., 2000 (includingsupplements through March 2002); Short Protocols in Molecular Biology,4^(th) Ed., Ausbel, Brent, and Moore, eds., John Wiley & Sons, New York,N.Y., 1999; Davis et al., Basic Methods in Molecular Biology, McGrawHill Professional Publishing, 1995; Molecular Biology Protocols (see thehighveld.com website), and Protocol Online (protocol-online.net).Exemplary gene products for genetically modifying the geneticallyengineered cells of the invention include, plasminogen activator,soluble CD-4, Factor VI II, Factor IX, von Willebrand Factor, urokinase,hirudin, interferons, including alpha-, beta- and gamma-interferon,tumor necrosis factor, interleukins, hematopoietic growth factor,antibodies, glucocerebrosidase, adenosine deaminase, phenylalaninehydroxylase, human growth hormone, insulin, erythropoietin, VEGF,angiopoietin, hepatocyte growth factor, PLGF, and the like.

In certain embodiments, the prevascularized construct further comprisesappropriate stromal cells, stem cells, Relevant Cells, or combinationsthereof. As used herein, the term “stem cells” is used in a broad senseand includes traditional stem cells, progenitor cells, preprogenitorcells, reserve cells, and the like. Exemplary stem cells includeembryonic stem cells, adult stem cells, pluripotent stem cells, neuralstem cells, liver stem cells, muscle stem cells, muscle precursor stemcells, endothelial progenitor cells, bone marrow stem cells,chondrogenic stem cells, lymphoid stem cells, mesenchymal stem cells,hematopoietic stem cells, central nervous system stem cells, peripheralnervous system stem cells, and the like. Descriptions of stem cells,including method for isolating and culturing them, may be found in,among other places, Embryonic Stem Cells, Methods and Protocols,Turksen, ed., Humana Press, 2002; Weisman et al., Annu. Rev. Cell. Dev.Biol. 17:387-403; Pittinger et al., Science, 284:143-47, 1999; AnimalCell Culture, Masters, ed., Oxford University Press, 2000; Jackson etal., PNAS 96(25):14482-86, 1999; Zuk et al., Tissue Engineering,7:211-228, 2001 (“Zuk et al.”); Atala et al., particularly Chapters33-41; and U.S. Pat. Nos. 5,559,022, 5,672,346 and 5,827,735.Descriptions of stromal cells, including methods for isolating them, maybe found in, among other places, Prockop, Science, 276:71-74, 1997;Theise et al., Hepatology, 31:235-40, 2000; Current Protocols in CellBiology, Bonifacino et al., eds., John Wiley & Sons, 2000 (includingupdates through March, 2002); and U.S. Pat. No. 4,963,489. The skilledartisan will understand that the stem cells and/or stromal cellsselected for inclusion in a prevascularized construct are typicallyappropriate for the intended use of that construct.

The term “Relevant Cells”, as used herein refers to cells that areappropriate for incorporation into a prevascularized construct, based onthe intended use of that construct. For example, Relevant Cells that areappropriate for the repair, restructuring, or repopulation of damagedliver may include, without limitation, hepatocytes, biliary epithelialcells, Kupffer cells, fibroblasts, and the like. Exemplary RelevantCells for incorporation into prevascularized constructs include neurons,myocardiocytes, myocytes, chondrocytes, pancreatic acinar cells, isletsof Langerhans, osteocytes, hepatocytes, Kupffer cells, fibroblasts,myocytes, myoblasts, satellite cells, endothelial cells, adipocytes,preadipocytes, biliary epithelial cells, and the like. These types ofcells may be isolated and cultured by conventional techniques known inthe art. Exemplary techniques can be found in, among other places, Atalaet al., particularly Chapters 9-32; Freshney, Culture of Animal Cells AManual of Basic Techniques, 4th ed., Wiley Liss, John Wiley & Sons,2000; Basic Cell Culture: A Practical Approach, Davis, ed., OxfordUniversity Press, 2002; Animal Cell Culture: A Practical Approach,Masters, ed., 2000; and U.S. Pat. Nos. 5,516,681 and 5,559,022.

The skilled artisan will appreciate that such stromal cells, stem cells,and/or Relevant Cells may be incorporated into the prevascularizedconstructs during or after preparation. For example, but not limited to,combining microvessel fragments, stem cells, Relevant Cells, and/orstromal cells in a liquid three-dimensional culture, such as collagen,fibrin, or the like, or seeding or sodding stem cells, Relevant Cells,and/or stromal cells in or on a prevascularized construct may beachieved. Exemplary combinations of appropriate stem cells, stromalcells, and Relevant Cells for incorporation into prevascularizedconstructs include: islets of Langerhans and/or pancreatic acinar cellsin a prevascularized construct for revascularizing a damaged pancreas;hepatocytes, hepatic progenitor cells, Kupffer cells, endothelial cells,endodermal stem cells, liver fibroblasts, and/or liver reserve cells ina prevascularized construct for revascularizing a damaged liver Forexample, but not limited to, appropriate stem cells or stromal cells fora prevascularized construct for vascularizing, repairing, andreconstructing a damaged or disease liver might comprise liver reservecells, liver progenitor cells, such as, but not limited to, liverfibroblasts, embryonic stem cells, liver stem cells; cardiomyocytes,Purkinje cells, pacemaker cells, myoblasts, mesenchymal stem cells,satellite cells, and/or bone marrow stem cells for revascularizing adamaged or ischemic heart (see, e.g., Atkins et al., J. of Heart andLung Transplantation, December 1999, at pages 1173-80; Tomita et al.,Cardiovascular Research Institute, American Heart Association, 1999, atpages 92-101; Sakai et al., Cardiovascular Research Institute, AmericanHeart Association, 1999, at pages 108-14); and the like.

II. Methods for Vascularizing Engineered Tissues and Organs

In certain embodiments, methods are provided for vascularizingengineered tissues comprising combining at least one prevascularizedconstruct with an engineered tissue to produce a vascularized engineeredtissue. In certain embodiments, prevascularized constructs forvascularizing engineered tissues further comprise at least one stromal,stem cell, Relevant Cell, or genetically engineered cell. In certainembodiments, prevascularized construct for vascularizing engineeredtissues comprise at least one cytokine, chemokine, antibiotic, drug,analgesic, anti-inflammatory, or the like. Methods for preparingengineered tissues are well known in the art. Descriptions of suchtechniques may be found in, among other places, Atala et al.; Lanza etal.; Masters; and in U.S. Pat. Nos. 4,963,489; 5,266,480; 5,510,254;5,512,475; 5,516,680; 5,516,681; 5,518,915; 5,541,107; 5,578,485;5,624,840; 5,763,267; 5,785,964; 5,792,603; 5,842,477; 5,858,721;5,863,531; 5,902,741; 5,962,325; 6,022,743; 6,060,306; 6,121,042; and6,218,182.

According to certain methods for vascularizing engineered tissues, theterm “combining” comprises placing or implanting at least oneprevascularized construct on any surface of, within, between layers of,or adjacent to, said engineered tissue. In certain embodiments,combining comprises coating the engineered tissue with a prevascularizedconstruct. For example, but without limitation, an engineered tissue isdipped into a liquid prevascularized construct or a liquidprevascularized construct is poured or sprayed on an engineered tissue.In certain embodiments, such liquid prevascularized construct coatingthe engineered tissue is polymerized. In certain embodiments, suchcoated engineered tissues are incubated prior to implantation into arecipient animal or human. In certain embodiments, the prevascularizedconstruct is combined with the engineered tissue by injection. Incertain embodiments, such injected construct polymerizes in situ,following injection. In certain embodiments, such injectedprevascularized construct comprises at least one cultured microvesselconstruct, at least one freshly isolated microvessel construct, or both.

In certain embodiments, combining at least one prevascularized with anengineered tissue comprises attaching at least one prevascularizedconstruct to at least one engineered tissue, using techniques known inthe art. Exemplary attachment means include suturing, stapling, forexample, with surgical staples, glue or adhesive, such as surgical glue,biochemical interactions such as with the extracellular matrix,photo-activated glue, fibrin glue, acrylate-based adhesives, and thelike.

In certain embodiments, combining comprises placing the at least oneprevascularized construct between the layers of an engineered tissue,such that at least one surface of at least one prevascularized constructis adjacent to, or in contact with, at least one surface of at least oneengineered tissue. In certain embodiments, combining comprises insertingor implanting at least one prevascularized construct within anengineered tissue, for example, but not limited to, within a designedpocket, bore, crevice, or the like. In certain embodiments, theprevascularized construct is inserted within an incision in theengineered tissue. In certain embodiments, combining comprises wrappingat least one prevascularized construct around or within at least oneengineered tissue, such that the prevascularized construct envelopes orsubstantially envelopes the engineered tissue, or is enveloped orsubstantially enveloped by the engineered tissue. In certainembodiments, combining comprises forming or incorporating at least oneprevascularized construct into the engineered tissue during the tissueengineering process. In certain embodiments, combining comprisesculturing at least one prevascularized construct on or within a growingengineered tissue during the tissue engineering process, such as in abioreactor. In certain embodiments, at least one prevascularizedconstruct is enveloped or substantially enveloped by the adjacent tissueor organ during the tissue engineering process.

In certain embodiments, the combined engineered tissue and at least oneprevascularized construct are incubated, for example within a bioreactoror humidified incubator, prior to in vivo implantation into a recipientanimal or human. In certain embodiments, combining comprises implantingat least one engineered tissue comprising at least one prevascularizedconstruct directly into a recipient animal or human with little or noadditional incubation.

In certain embodiments, the implanted prevascularized construct servesas a nucleation site for vascularizing the engineered tissue. In certainembodiments, appropriate stromal cells, stem cells, and/or RelevantCells from the prevascularized construct will support the integration ofthe engineered tissue within the recipient animal or human. Constructscomprising genetically engineered cells may produce recombinant productsthat are distributed systemically via the bloodstream or delivered tothe local microenvironment to induce repair, wound healing, or the like.

III. Methods for Revascularizing Damaged or Injured Tissues or Organs

In certain embodiments, methods for revascularizing damaged or injuredtissues or organs, i.e., tissues or organs in need of revascularizationand repair or reconstruction, are provided. In certain embodiments,prevascularized constructs for revascularizing tissues or organs furthercomprise at least one appropriate stromal cell, stem cell, RelevantCell, or genetically engineered cell. In certain embodiments,prevascularized constructs for revascularizing tissues or organscomprise at least one cytokine, chemokine, antibiotic, drug, analgesic,anti-inflammatory, or the like. In certain embodiments, theprevascularized construct, once implanted in vivo, will develop afunctional vascular bed and inosculate with the surrounding functionalvascular system and perfuse, or be capable of perfusing, the damagedtissue or organ.

According to certain methods for revascularizing tissues or organs, atleast one prevascularized construct is combined with said tissue ororgan and a revascularized tissue or organ is generated. According tocertain methods for revascularizing tissues or organs, the term“combining” comprises placing or implanting at least one prevascularizedconstruct on any surface of, within, between the layers of, or adjacentto, said tissue or organ. In certain embodiment, the prevascularizedconstruct is implanted in the tissue or organ by injection. In certainembodiments, such injected construct will polymerize in situ, followingimplantation. In certain embodiments, such injected prevascularizedconstruct comprises at least one cultured microvessel construct, atleast one freshly isolated microvessel construct, or both. In certainembodiments, combining comprises attaching at least one prevascularizedconstruct to at least one tissue or organ in need of revascularizing,using techniques known in the art, such as described above.

The skilled artisan understands that certain tissues and organs arecovered by or contain a layer of fibrous tissue, connective tissue,fatty tissue, or the like, and that the underlying tissue or organ canbe revascularized without removing this layer. Such a layer may benaturally occurring (such as a serosal layer, mucous membrane, fibrouscapsule, or the like), may result form fibrosis, necrosis, or ischemia,due to disease, defect, injury, or biochemical deficiency. Typically,the microvessel fragments of the prevascularized construct can penetratesuch a layer and inosculate with the vasculature of the underlyingtissue or organ, revascularizing the tissue or organ. Thus, combiningthe prevascularized construct with the tissue or organ in need ofrevascularization, comprises placing the prevascularized construct on orin such layer. For example, but not limited to, placing theprevascularized construct on: the meninges to revascularize braintissue; the epicardium to revascularize the myocardium; the peritoneumand/or serosa, to revascularize portions of the large intestine; theconjunctiva and/or subconjunctiva to revascularize the eye; the trachealsurface to revascularize the trachea; the bucchal mucosa torevascularize the mouth; the pleural and/or serosal surface torevascularize the lung; the pleural and/or peritoneal surface torevascularize the diaphragm; the skin to revascularize non-healing skinulcers, such as diabetic ulcers; the pericardial surface torevascularize the pericardium; and the like.

In certain embodiments, the prevascularized construct, when combinedwith the tissue or organ within the animal or human, will developfunctional vascular bed and inosculate with the surrounding functionalvascular system and perfuse the damaged tissue or organ. In certainembodiments, the implanted prevascularized construct serves as anucleation site for revascularizing the damaged tissue or organ. Incertain embodiments, appropriate stem cells, stromal cells, and/orRelevant Cells from the prevascularized construct will support therestructuring and repair of the damaged tissue or organ. Constructscomprising genetically engineered cells may produce recombinant productsthat are distributed systemically via the bloodstream or delivered tothe local microenvironment to induce repair, wound healing, or the like.

The invention, having been described above, may be better understood byreference to examples. The following examples are intended forillustration purposes only, and should not be construed as limiting thescope of the invention in any way.

EXAMPLES Example 1

We have developed a microvascular construct consisting of culturedmicrovessel in a 3-dimensional collagen matrix. The construct isprepared from rat-derived, freshly isolated, intact microvesselfragments (arterioles, capillaries and venules), which are subsequentlycultured in a collagen I matrix. After 7-10 days, these fragments expandinto an extensive, patent 3-dimensional capillary like networkcontaining both endothelial cells and mural cells. Subcutaneoustransplantation of mature microvascular constructs into SCID mice for 14days resulted in the perfusion of blood within vessels of the constructas detected by laser-Doppler perfusion imaging (LDPI), and OrthoganolPolarization Spectroscopy (OPS). The presence of RBCs within vessels wasconfirmed by hemotoxylin and eosin (H&E) staining. Control collagen gelslacking microvessel remained avascular and partially degraded.Evaluation of transplanted constructs over time revealed blood flowwithin vessels as early as 3 days post-transplantation. These resultssuggest that prevascularized tissue constructs may accelerate theestablishment of blood perfusion through the construct followingtransplantation.

Example 2 Rat Microvessel Isolation and Preparation of CulturedMicrovessel Constructs

Rat fat microvessel fragments (RFMF) were isolated from the epididymalfat pads (8 to 10 milliliters (mLs)) of retired breeder Sprague Dawleyor Fischer 344 rats, essentially as described (Carter et al., Surgery,120:1089-94, 1996). Harvested fat pads were washed in EFAF-PBS(Dulbecco's cation-free phosphate buffered saline (DCF-PBS) supplementedwith 0.1% essentially fatty acid free BSA (EFAFBSA; fraction V Sigma,St. Louis, Mo.), finely minced, placed in an Erlenmeyer flask containinga stir bar, and digested in PBS supplemented with 2 mg/mL collagenase(Worthington Biochemicals) and 2 mg/mL bovine serum albumin (BSA) for 10minutes at 37° C. with shaking for mechanical-assisted enzymaticdisruption. The solution was placed in a room temperature centrifuge andthe microvessel fragments were pelleted at 700×g for 3 minutes. Vesselfragments were transferred to 15 or 50 mL polypropylene conical tubes(Falcon), washed using approximately 12 mLs of EFAF-PBS and separatedfrom adipose cells by centrifugation in an IEC tabletop centrifuge at600-700×g for 3 minutes. Following centrifugation the fat cake wasremoved by decanting and the pelleted microvessel fragments weresuspended in 12 ml EFAF-PBS. Tissue debris and large vessel pieces wereremoved by filtering through a nylon screen of 500:m pore size. RFMFwere collected from the filtrate by screening the filtrate through anylon screen of 30 micron pore size. The RFMF were collected from thescreen, placed in 15 mL polypropylene conical tubes, washed twice bypipetting using approximately 12 mLs of EFAF-PBS per wash, andcentrifuged as before.

A nonpolymerized collagen solution was prepared by mixing theappropriate volume of stock rat tail collagen I stock (4 mg/ml) in 0.2NHCl (BD Biosciences, Bedford, Mass.) with 4× concentration of DMEMculture medium (Gibco BRL) to produce a final concentration of 3 mg/mlcollagen in DMEM. This solution was placed on ice and the pH wasneutralized by adding approximately 12 μL of 1 M NaOH per ml of collagensolution. The pH indicator in the DMEM changed from yellow (acidic) tored (neutral). Final collagen concentrations of greater than 1.5 mg/mLand less than 4 mg/mL produce robust microvessel fragment growth andangiogenesis.

The washed RFMF were counted using phase contrast microscopy andresuspended in the liquid collagen solution at a concentration ofapproximately 12,000-15,000 RFMF/mL. This liquid prevascularizedconstruct was plated into wells of 48-well tissue culture plates andplaced in a humidified, 37° C. incubator for 15-30 minutes to polymerizethe construct. When the prevascularized construct was polymerized, anequal volume (approximately 0.2 mL) Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% fetal bovine serum (10% FBS-DMEM) was addedto each well. The plates were incubated in a humidified 37° C., 5% CO₂incubator and the microvascular networks in these cultured microvesselconstructs were allowed to develop within the culture well for seven toten days.

Human fat microvessel fragments (HFMF) were also isolated and culturedmicrovessel constructs were prepared from human abdominoplasty followingthis procedure. HFMF can also be isolated from liposuction fat accordingto this procedure except that 4 mg/mL collagenase is used in theenzymatic disruption step.

The person of ordinary skill will understand that freshly isolatedmicrovessel constructs may also be produced following this procedure,except that the constructs are not incubated for 7-10 days in anincubator. In order to get a solidified collagen gel construct, however,freshly isolated microvessel constructs would typically be incubated ata temperature sufficient to allow polymerization of the collagen.

The skilled artisan will appreciate that other enzymes, such as dispase,trypsin, elastase, liberase, or the like, may be used in such digestionsteps in place of collagenase. The skilled artisan will also understandthat many such digestive enzyme preparations will vary in activity fromvendor to vendor and from lot to lot (see, e.g., London et al., Diabetes& Metabolism, 23:200-07, 1998). For example, a given enzyme lot mighthave a high specific activity such that its use at the identifiedconditions will result in RFMF preparations that consist of anunacceptably low number of microvessel fragments due to over-digestionof the preparation. Further, certain enzyme preparations reportedlycontain various contaminants that may be harmful to tissues, such asendotoxins and additional contaminating proteases (see, e.g., Arita etal., Pancreas 23:62-67, 2001). Thus, the person of ordinary skillunderstands that one routinely titrates each enzyme lot in a standardassay to determine its comparative enzyme activity prior to use.

Example 3 Implantation of Prevascularized Constructs to RevascularizeIschemic Heart Tissue

Prevascularized constructs comprising RFMF were prepared essentially asdescribed in Example 2, above. Cultured microvessel constructs werecultured in 48-well plates with 10% FBS-DMEM for seven days in ahumidified 37° C., 5% CO₂ incubator prior to implantation. During thisculture period the microvessels formed an extensive microvessel networkthroughout the three-dimensional culture (see FIG. 1). Freshly isolatedmicrovessel constructs were prepared and then immediately implanted.

To demonstrate the revascularization of ischemic myocardium, acryoinjury model of infarction was used to generate infracted cardiactissue. Male retired breeder Fischer 344 rats were used for theepicardial implantation of the prevascularized constructs. Epicardialaccess was gained through a lateral thoracotomy and cryoinjury wasachieved by administration of a metal probe that had been cooled inliquid nitrogen on the anterior free wall of the left ventricle.Following infarct formation, prevascularized constructs, either freshlyisolated microvessel constructs or cultured microvessel constructs, wereattached directly to the epicardial surface, over the site of inducedinjury, using two 8-0 sutures. Control rats received infarct only, orinfarct followed by the implantation of a three-dimensional culturelacking RFMF. The chest was closed in layers and the animals recovered.The animals were sacrificed and their hearts were excised 14 dayspost-implantation.

Tissue sections were fixed in HISTOCHOICE™ (Amresco, Solon, Ohio) andparaffin embedded for histology and immunochemistry according toconventional methods. Six micron sections were cut for standard H&Estaining as well as for endothelial cell identification using the GS-1lectin (Griffonia simplicifolia). H&E staining confirmed the presence ofa mature, patent vasculature comprised of arterioles, venules andcapillaries throughout both the explanted freshly isolated microvesselconstructs and the explanted cultured microvessel constructs.

Gross observation of the explanted prevascularized constructs revealedthe presence of microvessels within them by 14 days post-implantation.The constructs were well integrated with the epicardial surface andvascularization was seen throughout both the freshly isolatedmicrovessel constructs and the cultured microvessel constructs. Thepresence of red blood cells within vessel walls was noted throughout theprevascularized constructs, demonstrating that the implantedprevascularized constructs rapidly inosculated with the underlyingepicardial vasculature. Control implants remained avascular. EpicardialVascular Densities were higher in the animals receiving prevascularizedconstructs than animals receiving control constructs.

The skilled artisan will understand that prevascularized constructs canbe combined with tissues or organs by implanting the constructs atappropriate anatomical sites, following similar procedures. For example,prevascularized constructs may be combined with damaged or diseasedtissues or organs in need of revascularization, by placing theconstructs on the surface of, within, adjacent to, or on an externallayer of the damaged or diseased tissue or organ. The skilled artisanwill also understand that prevascularized constructs can be combinedwith tissues, organs, or engineered tissues by injection into, oradjacent to, a damaged or diseased tissues or organs or engineeredtissue, for example, a syringe with a needle of an appropriate gauge toallow the microvessel fragments and additional cell types (if present)to pass through without being damaged.

Example 4 Implantation and Evaluation of Cultured Microvessel Constructs

Cultured microvessel constructs, comprising RFMF, such as described inExample 2 were cultured for 7-11 days. During the first 5 days ofculture prior to implantation, angiogenic sprouts were observed onindividual fragments in the prevascularized constructs. These sproutswere seen to undergo a dynamic process of growth resulting in a loosecollection of elongated, simple microvessels by day 11 (FIG. 4). Thecultured prevascularized constructs and control constructs lackingmicrovessel fragments (“control constructs”) were implanted in thesubcutaneous position on the flanks of SCID (Severe CombinedImmuno-Deficient) mice for 1, 3, 5, 7, 10, 14, 21, 28, or 35 days or for4 months. Each mouse received two implants, a prevascularized constructon one side and a control construct on the other.

At the time of explant, implants were observed for the presence ofblood-containing vasculature and photographed. Explants were fixed in 2%paraformaldehyde/PBS and paraffin embedded for histology. Generalhistological structure was determined on 6 μm thick sections withhematoxylin and eosin (H&E) staining and the vasculature identifiedusing a rodent-specific lectin, GS1 (Griffonia simplicifolia).

In contrast to the implanted control constructs, the implantedprevascularized constructs were associated with superficial,blood-filled vessels (FIG. 5). Orthogonal polarized spectral (OPS)imaging, which selectively detects hemoglobin, revealed blood-containingvessel structures throughout the constructs as early as 3 dayspost-implantation (FIG. 5). Between days 3 to 14 post-implantation, thesimple network was observed to remodel into a more typical appearingvasculature with vessels of various dimensions and orientations presentthroughout the implant (FIG. 5). This mature vessel architecture wasobserved to be established by day 7 and persisted through at least day35. Only surface blood due to the dissection was detected on implantedcontrol constructs with OPS (FIG. 5). Histology of explanted,prevascularized constructs (FIG. 6) confirmed the presence of blood inthe vessels and the heterogeneous, more mature vascular networkindicated by the OPS imaging. A full range of vessel types commonly seenin a mature, functional vascular bed was observed, including smallarteries, arterioles, capillaries, venules and veins (FIG. 6). Based onthe histology, it was evident that blood filled vessels were present inimplants as early as day 1 post-implantation (FIG. 6). However, vesselperfusion was limited to the implant periphery, adjacent to hosttissues.

Staining for an endothelial specific marker with the lectin GS-1verified the vascular nature of the vessels (FIG. 7) as well as revealeda limited number of non-vascular, single cells (GS-1 negative) dispersedthroughout the implant (FIG. 7). These dispersed, single cells were ofrat origin (see below) and thus were part of the original isolate usedto form the prevascularized construct. Control constructs were found tobe void of vessels or cells. Histology sections of implants stained forthe presence of macrophages revealed limited inflammatory cellinfiltration into the implant (FIG. 7).

Since the prevascularized constructs comprising RFMF from male rats wereimplanted in female SCID mice, it was possible to determine by PCR ifthe vessels within the prevascularized construct were derived from theimplanted cultured microvessel construct or due to the ingrowth of host,mouse vessels. Rat genomic DNA was isolated from rat-tail tissue, rat Ychromosome DNA was synthesized by PCR (F: ggt tct aga ctg taa aac ccagac R: act taa aac taa gct tat tgg cca) and labeled with biotin (VectorLaboratories). Eight micron explant sections were deparaffinized inxylene, rehydrated in an ethanol series, treated with 0.2 M HCl,followed by an incubation in 0.1% TritonX-100 for 2 min and finallytreated with 10 μg/ml of proteinase K in PBS. Following post-fixationwith 4% paraformaldehyde, chromosomal DNA was released by treatment with0.1 M TEA, 0.25 M acetic anhydride and washed 2 times in 2×SSC. Sectionswere dehydrated in ethanol from 50% to 100% and hybridized.

A biotin-labeled DNA probe, specific to a repeat element of the rat Ychromosome (Essers et al., Cytogenet. Cell. Genet. 69:246-252, 1995),was used to analyze sections from day 5 and day 28 implants. Sectionswere pre-hybridized in 50% formamide, 20% dextran sulfate in 2×SSC, andsalmon sperm DNA for 20 minutes at 55°. This Y-chromosome probe wasdenatured at 95° C. for 5 minutes and was incubated overnight inpre-hybridization solution at 42° C. The hybridized sections were washedtwice for five minutes with 4×SSC 5 minutes at room temperature, for 20minutes in 2×SSC at room temperature, for 15 minutes in 0.2×SSC at 42°C., and for 15 minutes in 0.1×SSC at 42° C. The hybridized labeled probewas detected via an enzyme-conjugated StrepAvidin according tomanufacturer's instructions (Vector Laboratories).

As seen in FIG. 8, vessel-like structures and single cells throughoutthe implant were detected with the Y chromosome-specific probe. Cellswithin the implants at day 5 and day 28 were predominately Ychromosome-positive cells (FIG. 8). No Y-positive cells were observedwithin the underlying muscle of the host mouse (FIG. 8).

Freshly isolated microvessel constructs were also prepared using RFMF,as described in Example 2, and implanted subcutaneously into the flanksof female scid/scid mice, as described above. As with culturedmicrovessel constructs, the freshly isolated microvessel constructsdeveloped a recognizable, blood-filled vasculature (FIG. 9). Freshlyisolated microvessel constructs were also prepared as described inExample 2, except that human microvessel fragments isolated fromabdominoplasty tissues were used instead of RFMF. These constructs wereimplanted subcutaneously into the flanks of female scid/scid mice, asdescribed above. As with the RFMF microvessel constructs, implantedprevascularized constructs comprising human microvessel fragments werewell perfused by day 7 post-implantation. Histology sections of theseexplanted constructs were probed with the human endothelialcell-specific lectin, UAE (Ulex Europaeus Agglutinin I), to verify thehuman origin of the implant vasculature (FIG. 9).

Example 5 Isolation of Human Stem Cells from Liposuction Fat

Multilineage progenitor cells, believed to be stem cells, are isolatedfrom human adipose tissue, essentially as described by Zuk et al. (seealso Hauner et al., J. Clin. Endocrinol. Metabol. 64:832, 1987; Katz etal., Clin. Plast. Surg. 26:587, 1999). Human adipose tissue is obtainedfrom a liposuction procedure using a hollow blunt-tipped cannulaintroduced into the subcutaneous space through small incisions ofapproximately 1 centimeter (cm). The cannula is attached to gentlesuction and moved through the adipose compartment to mechanicallydisrupt the fat tissue. A solution of saline and the vasoconstrictorepinephrine is infused into the adipose compartment to minimize bloodloss and contamination of the fat tissue by peripheral blood cells.Approximately 300 cubic centimeters (cc) of the lipoaspirate isextensively washed with equal volumes of PBS and the washed lipoaspirateis digested using a 0.075% collagenase solution for 30 minutes at 37° C.Enzyme activity is neutralized using 10% FBS-DMEM and the preparation iscentrifuged at 1200×g to obtain the high density pellet, referred to asthe stromal vascular fraction (SVF). Contaminating red blood cells arelysed by resuspending the SFV pellet in 160 mM NH₄Cl and incubating themixture at room temperature for ten minutes. The treated SFV iscentrifuged at 1200×g and the resuspended pellet is filtered through a100 μm nylon mesh to remove cellular debris. The SFV is suspended in 10%FBS-DMEM supplemented with 1% antibiotic/antimycotic solution, platedinto multi-well plates, and incubated in a humidified incubator at 37°C. and 5% CO₂. Following overnight incubation, the plates are washedextensively with PBS to remove nonadherent red cells. The resultingprocessed lipoaspirate (PLA) can reportedly be maintained in 10%FBS-DMEM in a 5% CO₂ incubator at 37° C. for at least 13 passages (165days in culture) without a significant loss in population doubling overtime.

Reportedly, cells in this PLA undergoes lineage-specific differentiationunder appropriate conditions in vitro, as described by Zuk et al. Thismultilineage potential reportedly includes adipogenic, osteogenic,chondrogenic, and myogenic lineages. Thus, the skilled artisan willappreciate that PLA is a readily available source of multilineage cells,presumably stem cells, that would be appropriate for inclusion inprevascularized constructs for combining with a variety of organs andtissues, including engineered tissues. PLA cells can be incorporatedinto prevascularized constructs by mixing them into thethree-dimensional culture environment along with microvessel fragments,following, for example, the procedure described in Example 2 above. Inaddition, PLA can be implanted in parallel with, or in addition to, theprevascularized constructs. Once in the appropriate environment in vivo,the multilineage/stem cells in PLA will differentiate into theappropriate lineage to allow repopulation or reconstruction of damagedor dysfunctional tissue or organs in need of revascularization.

The skilled artisan will understand that the same liposuctionpreparation may be used to isolate both microvessel fragments and PLAand that these cells may be for used in preparing freshly isolatedmicrovessel constructs or cultured microvessel constructs. Further, anautologous construct can be prepared using, for example, but withoutlimitation, PLA and microvessel fragments obtained from the patient'slipoaspirate and a fibrin-based scaffold derived from the patient'sblood.

The skilled artisan will also understand that stem cells from numerousother sources may be used in various embodiments of the invention in ananalogous fashion to that described in this example. For example, butnot limited to, embryonic stem cells and mesenchymal stem cells obtainedfrom bone marrow aspirate according to conventional techniques (e.g.,Liechty et al., Nature Medicine 6(11):1282-1286, 2000) or fromcommercially available sources (e.g., Clonetics, Walkersville, Md.).

The skilled artisan will appreciate that the effective concentration ofeach additional cell type (e.g., stem cells, stromal cells, RelevantCells) within the prevascularized construct is dependent on the celltype and the intended use of the prevascularized construct. Thus, theperson of ordinary skill will understand that it is routine to titrateeach cell type in test prevascularized constructs to identify theeffective concentration for a particular use. For example, to determinethe effective concentration of PLA-derived human stem cells inprevascularized constructs, test constructs prepared according themethod of Example 2 could be prepared as follows. Eighteen parallelliquid three-dimensional culture preparations comprising 13,000 humanmicrovessel fragments each and either 0, 10, 100, 1000, 10000, or 50,000PLA-derived human stem cells/ml are prepared in six triplicate sets, andallowed to polymerize. The 18 parallel constructs are combined withtarget tissues or organs by implanting the constructs directly into testanimals, as described in Examples 3 and 4. After an appropriateimplantation period, the prevascularized constructs would be explantedand the recipient animal, the implant, and the relevant tissue or organwould be evaluated, as described in Examples 3 and 4. To evaluate theeffect of the additional cell type(s) on the proliferation and growthmicrovessel fragments in cultured microvessel constructs, similartriplicate constructs could also be incubated, for example in ahumidified 37° C., 5% CO₂ incubator, and evaluated over a seven to tenday period, as described in Example 4.

The skilled artisan will understand that further refinement of theappropriate number of additional cells for a particular prevascularizedconstruct can be determined by additional experiments, based on theresults of the above procedure. For example, if in the first experimentthat 1000 additional cells/ml demonstrated the best results, additionaltests using 500, 2000 and 6000 cells/ml would allow further refinementof the optimal number of additional cells per construct. A similarprocedure could be followed to determine the appropriate concentrationof any stem cell, stromal cell, Relevant Cell, genetically engineeredcell, or combinations thereof, in a prevascularized construct.

Example 6 Isolation of Human Microvessel Endothelial Cells fromLiposuction Fat

Liposuction fat was procured from the procedure site and transported inmedium, for example, M199. The fat in M199 was poured over a sterilesieve with a 500 micron pore size and the retentate was washed withDCF-PBS to remove red blood cells. Approximately 10 grams (about 10 mLs)of the washed liposuction fat was transferred to a sterile 50 mLpolycarbonate Erlenmeyer flask containing a TEFLON® coated stir bar andten mLs of sterile collagenase solution (4 mg/ml colagenase Pure 1(Worthington Biochemicals) and 4 mg/ml human serum albumin in DCF-PBS)was added. The flask was stirred at approximately 64 revolutions perminute for 30 minutes in a Dubnoff water bath 37° C. to digest the fat.The digested solution was transferred to 15 mL polypropylene conicalcentrifuge tubes and centrifuged for four minutes at 700×g. Theendothelial cells and red blood cells pelleted, the fat formed a “plug”at the top of the tube with the collagenase solution supernatant wasbetween them.

The fat and supernatant were discarded, the cell pellet was resuspendedin approximately 2 mLs DCF-PBS supplemented with 0.1% BSA, and thentransferred into clean 15 mL conical centrifuge tubes, taking care thatthe transfer of fat was avoided. The tubes were centrifuged at 700×g forthree minutes at room temperature. The supernatant was poured off andthe endothelial cell preparation was resuspended in sodding medium (M199supplemented with between 0.1% and 1% EFAFBSA). (See also, Stopeck etal., Cell Transplant. 6:1-8, 1997; Hoying et al., J. Cell. Physio.168:294-304, 1996).

Example 7 Generation of Genetically Engineered Endothelial Cells forIncorporation into Prevascularized Constructs

Human endothelial cells are genetically engineered to generate cellsthat constitutively express human gamma interferon (γ-IFN), essentiallyas described by Stopeck et al., Cell Transplantation 6:1-8, 1997. Thehuman endothelial cell pellet from Example 6 is resuspended in M199supplemented with 20% heat-inactivated FBS, 5 mM HEPES, 1.7 mML-glutamine, and 60 μg/mL endothelial cell growth supplement (Jarrell etal., J. Vasc. Surg. 1:757-64, 1984) containing 25 μg/mL heparin andplated on gelatin coated polystyrene T-25 tissue culture flasks andincubated in a conventional humidified 37° C., 5% CO₂ incubator andmaintained in culture.

Supernatants of high titer (1×10⁶−1×10⁷ cfu/mL) recombinant retroviruscontaining either the E. coli beta-galactosidase (β-gal) or human γ-IFNgene were obtained from Viagene, Inc. (San Diego, Calif.). Theserecombinant retroviruses comprise a Moloney murine leukemia virus genomewith viral structural genes replaced by either the β-gal or the humanγ-IFN gene. T-25 flasks of human endothelial cells at 30-40% confluencyare transduced for 6-18 hours on 2 consecutive days with mediacontaining 750 μg/mL protamine sulfate and retrovirus supernatants at amultiplicity of infection of 5.

Forty-eight hours after transduction, cells are fixed with 2%formaldehyde prior to staining with X-gal solution (5 mM potassiumferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, and 1 mg/L X-gal(Sigma, St. Louis, Mo.) in PBS overnight at 37° C. The transductionefficiency is calculated as the number of cells staining positive forβ-gal divided by the total number of cells counted. β-gal transduced orhuman γ-IFN transduced endothelial cells are selected using 1 mg/mL G418(Gibco BRL) selection medium.

Total RNA is extracted from transduced and control endothelial cellsusing Trizol (Gibco BRL) for RT-PCR analysis, as described. Humanendothelial cells transduced according to this procedure reportedlyproduce 80-130 pg/mL of human γ-IFN per 10⁵ cells after 24 hours inculture (see Stopeck et al., Cell Transplantation 6:1-8, 1997 and U.S.Pat. No. 5,957,972).

The skilled artisan will understand that replacement of the human γ-IFNor β-gal gene in these recombinant retrovirus vectors with alternategenes of interest requires only routine manipulation using techniquesgenerally known in the art. Thus, any number of genes of interest may betransduced into and expressed by endothelial cells following thisexemplary technique. The skilled artisan will also understand that,following techniques generally known in the art, a variety of mammaliancells can routinely be transduced or transfected to express virtuallyany gene product of interest (see, e.g., Twyman, Advanced MolecularBiology: A Concise Reference, Bios Scientific Publishers, SpringerVerlag New York, particularly Chapter 24). Particularly useful geneproducts of interest include, for example, but without limitation,cytokines, insulin, human growth hormone, plasminogen activator, solubleCD-4, Factor VIII, Factor IX, von Willebrand Factor, urokinase, hirudin,interferons, including alpha-, beta- and gamma-interferon, tumornecrosis factor, interleukins, hematopoietic growth factor, antibodies,glucocerebrosidase, adenosine deaminase, phenylalanine hydroxylase,human growth hormone, insulin, erythropoietin, VEGF, angiopoietin,hepatocyte growth factor, PLGF, and other proteins or gene productsappropriate for local or systemic delivery, particularly blood-bornedelivery.

Genetically engineered cells, particularly genetically engineeredendothelial cells, may be incorporated into the prevascularizedconstructs of the invention at appropriate concentrations, as described.Prevascularized constructs comprising autologous microvessel fragmentsin an autologous three-dimensional culture matrix and geneticallyengineered cells prepared from autologous endothelial cells areparticularly useful for certain applications. The skilled artisan that awide variety of techniques may be used to genetically modify cells,i.e., transferring genes and nucleic acids of interest into recipientcells, using techniques generally known in the art, including, but notlimited to: transfection (e.g., the uptakeof naked nucleic acid), forexample, but not limited to polyethylene glycol transfection, chemicaltransfection (e.g., using calcium phosphate and DEAE dextran),lipofection, electroporation, direct injection, and microballistics; andtransduction, using a number of viral vectors, such as, withoutlimitation, adenovirus vectors, herpesvirus vectors, retrovirus vectors,including, but not limited to lentivirus vectors. Descriptions of suchtechniques may be found in, among other places, Ausubel et al., CurrentProtocols in Molecular Biology (including supplements through March2002), John Wiley & Sons, New York, N.Y., 1989; Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989; Sambrook and Russell,Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2001; Beaucage et al.,Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, NewYork, N.Y., 2000 (including supplements through March 2002); ShortProtocols in Molecular Biology, 4^(th) Ed., Ausbel, Brent, and Moore,eds., John Wiley & Sons, New York, N.Y., 1999; Davis et al., BasicMethods in Molecular Biology, McGraw Hill Professional Publishing, 1995;Molecular Biology Protocols (see the highveld.com website), ProtocolOnline (protocol-online.net); and Twyman, Advanced Molecular Biology: AConcise Reference, Bios Scientific Publishers, Springer-Verlag New York.

Example 8 Isolation of Pancreatic Islets

Human pancreatic islets are isolated using the tube method of ductalcanulation and collagenase infusion, essentially as described by Aritaet al., Transplantation 68(5):705-07, 1999. A polyethylene tube(INTRAMEDIC, Clay Adams, Parsippany, N.J.) with a tight-fitted injectionneedle on one end, a diameter of approximately 0.64 to 1.47 millimeters(mm) depending on the duct size, and of a length similar to the pancreasis used. The tube is inserted into the main duct of a whole pancreas,starting from the head and extending to the tail, and the duct isligated around the tube. The pancreas is infused and digested withapproximately 150-300 mL collagenase solution (3 mL/g pancreas weight),comprising 2-2.3 mg/mL of lots 9 or 522 collagenase P (BoehringerMannheim, Indianapolis, Ind.). The collagenase-infused pancreas ischopped into small pieces, placed in a digestion chamber with remainingcollagenase solution and gently agitated in a 37° C. water bath. Totalincubation time in collagenase is 15 minutes. The collagenase solutionis replaced by cold LAP-1 preservation solution (Islet Technology, NorthOaks, Minn.) and the digestion chamber is placed in an ice-water bathand gently agitated. The supernatant, containing islets and fragmentedacinar and duct tissue, is decanted every 5-10 minutes into collectionbottles containing LAP-1 solution and fetal bovine serum. Fresh LAP-1solution is added to the digestion chamber and cold digestion continuesuntil most of the islets are released, approximately 30-40 minutes. Thedigested tissues are collected and islets are purified by centrifugationon a discontinuous three-layer gradient of Euro-Ficoll solution using aCOBE2991 cell processor (COBE Laboratories, Lakewood, Colo.).

Numerous other islet purification techniques, generally known in theart, may also be employed. Exemplary islet purification techniques maybe found in, among other places, London et al., in Methods in CellTransplantation, Ricordi, ed., at pages 439-54, 1995; Lakey et al.,Transplantation 72:562-63, 2001; Olack et al., Human Immunol.60:1303-09, 1999; London et al., Diabetes Metab. 24:200-07, 1998;Linetsky et al., Diabetes 46:1120-23, 1997; Arita et al., Pancreas23:62-67, 2001; and Wang et al., Nat. Biotechnol. 15:358-62, 1997. Theperson of ordinary skill in the art will understand that such isolatedpancreatic islets may be useful as Relevant Cells in, for example,prevascularized constructs for revascularizing a damaged or diseasedpancreas.

Example 9 Isolation of Other Relevant Cells

Cells from animal or human liver are obtained as described by Macdonaldet al. in Atala et al., Chapter 11, particularly at pages 155-166. Humanadipocytes are obtained according to the method of Katz, as described inAtala et al., Chapter 20. Human smooth muscle cells are isolatedaccording to the method of Kim et al., as described in Atala et al.,Chapter 21. Human and animal cardiomyocytes are obtained using themethod of Soker et al., as described in Atala et al., Chapter 22 orSakai et al., Cardiovascular Research Institute, American HeartAssociation, 1999, at pages 108-14 (see also Tomita et al, Id. at pages92-101 (describing the use of bone marrow cells that differentiate intocardiomyocytes) and Atkins et al., J. of Heart and Lung Transplantation,1999, at pages 1173-80 (describing cellular cardiomyoplasty usingautologous skeletal myoblasts)). Myocytes, fibroblasts and satellitecells are obtained from animal or human striated muscle following themethods of Kosnick et al., as described in Atala et al., Chapter 23.Myoblasts are isolated and cultured according to the methods of Atkinset al., as described in J. of Heart and Lung Transplantation, 1999, atpages 1173-80. Chondrocytes from human or animal articular cartilage areobtained according to the methods of Kinner et al., as described inAtala et al., Chapter 25. Mouse and rat bone marrow cells are obtainedfrom femoral marrow and human bone marrow cells are obtained from bonemarrow aspirates or trabecular bone biopsies using the methods of Davieset al., as described in Atala et al., Chapter 26.

Relevant Cells isolated according to these or other conventional methodsknown in the art may be used in preparing prevascularized constructs ofthe invention. The skilled artisan will understand that in certainembodiments, suspensions of such Relevant Cells may be added to liquidthree-dimensional cultures in addition to microvessel fragments togenerate prevascularized constructs. In certain embodiments, suchRelevant Cells, the microvessel fragments, or both, are resuspended in aliquid three-dimensional culture matrix. Alternatively, such RelevantCells can be added to a scaffold or other preformed matrix before,after, or simultaneously with the microvessel fragments to formprevascularized construct according to the invention. Theseprevascularized constructs can then either be directly combined orcultured and then combined according to the methods disclosed herein,depending on whether freshly isolated microvessel constructs or culturedmicrovessel constructs are desired.

The person of ordinary skill will appreciate that prevascularizedconstructs may be prepared using Relevant Cells and microvesselfragments isolated from any mammalian species following the methodsdescribed herein. For example, rat prevascularized constructs can beprepared using rat microvessel fragments in an appropriate threedimensional culture matrix, with or without appropriate rat stem cells,rat stromal cells, rat hepatocytes, rat myocytes, etc. Similarly, arabbit prevascularized construct would comprise rabbit microvesselfragments, an appropriate three dimensional matrix, and optionallyrabbit stem cells, rabbit stromal cells, rabbit hepatocytes, rabbitmyocytes, etc.; and so forth. Such prevascularized constructs are thencombined with organs or tissues, including engineered tissues, accordingto the methods of the invention.

Example 10 Preparation of Prevascularized Constructs UsingFibrin-Derived Scaffolds

Three hundred milligrams of fibrinogen powder is dissolved in 10 mLHEPES-buffered saline solution (30 mg/mL) and passed through a0.2-micron syringe filter (fibrinogen solution). Two hundred and fiftyunits of thrombin powder is dissolved in 10 ml of HEPES-buffered saline(25 units/mL) and filtered through a 0.20-micron syringe filter(thrombin solution). The fibrinogen solution is diluted using Medium 199(M199) supplemented with 12% FBS to yield a diluted fibrinogen solutioncontaining 5 mg/mL fibrinogen and 10% FBS. The thrombin solution isdiluted with M199 containing 15 mM CaCl to yield a diluted thrombinsolution with a thrombin concentration of 2.5 units/mL. Four partsdiluted fibrinogen solution are mixed with one part of the dilutedthrombin solution and one part cell suspension and this mixture isplaced in an incubator at 37° C. to allow the fibrin to polymerize. Anacellular fibrin scaffold is prepared using an equal volume of mediumwithout cells in place of the cell suspension. The skilled artisan willappreciate that the concentration of cells or microvessel fragments inthe “cell suspension” must be adjusted to achieve the desired finalcell/microvessel fragment concentration, for example, approximately11,000-15,000 microvessel fragments per mL.

An autologous fibrin-derived three-dimensional culture or scaffold isprepared using materials obtained from a recipient patient's or animal'sblood as follows. Human or animal blood is collected in 9 mM bufferedsodium citrate. The citrate treated blood is centrifuged for 10 minutesat 300×g and the platelet-poor plasma supernatant is decanted. Fibringel formation is initiated by the addition of 50 mM CaCl₂ and theresulting fibrin suspension is incubated at 37° C. until the fibrinpolymerizes (see also, Williams et al., J. Surg. Res. 38:618-29, 1985;and Rupnick et al., J. Vascular Res. 9:788-95, 1989). The skilledartisan will understand that microvessel fragments may readily be addedto the fibrin suspension prior to polymerization or to the fibrin gel.Cell types and constituents, such as drugs, cytokines, analgesics, andthe like may also be added to the fibrin suspension or the fibrin gel.An allogeneic fibrin-derived three-dimensional culture may be preparedaccording to this Example, except that the fibrin is obtained, not fromthe intended recipient, but from another member of the same species.Additionally, the skilled artisan will appreciate that a fibrinsuspension comprising microvessel fragments may be used in, for example,but without limitation, preparing a vascularized engineered tissue, suchas in lieu of the nonpolymerized collagen solution described in Example11.

Example 11 Preparation of a Vascularized Engineered Tissue

A suspension comprising approximately 13,000 HFMF/mL, obtained fromliposuction fat according to Example 2, in the nonpolymerized collagensolution of Example 2, containing 2 ng/ml human VEGF₁₆₅ and 1 ng/mlhuman PDGF-BB (both from R&D Systems, Minneapolis, Minn.) is prepared. A5 cm×7 cm piece of polyglycolic acid (PGA) felt (Albany International)with a pore size ranging from 2-15 μm and one mm thick is placed in asterile glass pan. The suspension is gently poured into the glass panuntil the felt is covered forming a prevascularized construct. Theprevascularized construct is incubated at room temperature untilpolymerization occurs. A slice is made through the prevascularizedconstruct along the edge of the PGA felt using a sterile scalpel.

The prevascularized construct comprising the felt is gently removed fromthe dish and placed directly on top of a 5 cm×7 cm piece of freshlythawed DERMAGRAFT® human fibroblast-derived dermal substitute (AdvancedTissue Sciences, La Jolla, Calif.; see Atala et al., particularlyChapter 104). The prevascularized construct is attached to theDERMAGRAFT using one suture at each corner of the prevascularizedconstruct-engineered tissue composite. After trimming the composite tothe size of a debrided foot ulcer on the leg of a human patient withdiabetes, the composite is implanted into the wound bed on the patient.The composite is held securely in the wound bed using surgical dressingsand a vascularized engineered tissue forms.

Alternatively, an engineered tissue, for example, but not limited toengineered pancreatic tissue prepared in a bioreactor, for example, butwithout limitation, according to U.S. Pat. No. 6,022,743. A flexibleprevascularized construct comprising PGA felt is prepared according tothis Example. The engineered pancreatic tissue is removed from thebioreactor and combined with the flexible prevascularized construct bywrapping the flexible construct around the engineered pancreatic tissueand attaching it with 8-0 sutures. The prevascularizedconstruct-engineered pancreatic tissue combination is implanted in ahuman patient, following surgical following procedures. A vascularizedpancreatic tissue is generated in vivo.

The skilled artisan will understand, based on these illustrativeexamples, that flexible prevascularized constructs may be wrapped aroundor within engineered tissues, such as DERMAGRAFT, and implanted into ahuman and cultured to generate vascularized engineered tissue. Theskilled artisan will appreciate that prevascularized constructs may alsobe combined with an engineered tissue by placing the construct withinthe tissue. The combination is subsequently implanted and cultured invivo to generate vascularized tissue. Vascularized engineered tissuesmay also be prepared, for example, but without limitation, combining atleast one inflexible prevascularized constructs with an engineeredtissue, before or after the tissue is implanted in a human patient. Theskilled artisan will also appreciate that these techniques may be usedwith any engineered tissue to produce a vascularized engineered tissue.

Although the invention has been described with reference to variousapplications, methods, and compositions, it will be appreciated thatvarious changes and modifications may be made without departing from theinvention. The foregoing examples are provided to better illustrate theinvention and are not intended to limit the scope of the invention.

What is claimed is:
 1. A method for expanding substantially intact microvessel fragments construct adapted for use in vascularizing a tissue or an organ in a subject, said method comprising: a. isolating microvessel fragments; b. combining said isolated microvessel fragments with a liquid solution of biocompatible culture matrix to produce a solution of prevascularized construct comprising said isolated microvessel fragments; and c. culturing and expanding said microvessel fragments in said step (b) under conditions sufficient to produce a three dimensional culture matrix of prevascularized construct comprising an expanded microvessel fragments that is adapted for use in vascularizing a tissue or an organ of said subject.
 2. The method of claim 1, wherein said three dimensional culture matrix comprises at least one component selected from the group consisting of collagen, a fibrin, a gelatin, an alginate, a hydrogel, a dextran, a chemically-crosslinkable dextran, a photo-crosslinkable dextran, ePTFE, PTFE, PGA, PLA, PLGA, PET, a nylon, a vinyl, a silk, and a combination thereof.
 3. The method of claim 1 further comprising the steps of adding one or more components selected from the group consisting of at least one chemokine, at least one antibiotic, at least one drug, at least one analgesic agent, at least one anti-inflammatory agent, at least one immunosuppressive agent, and combinations thereof in or after any of steps (b) or (c).
 4. The method of claim 1, wherein said microvessel fragments are cultured for 7-11 days.
 5. The method of claim 1 further comprising the step of adding a cytokine in or after any of steps (b) or (c).
 6. The method of claim 5, wherein said cytokine comprises VEGF, PDGF, or a combination thereof.
 7. The method of claim 1 further comprising the step of adding one or more stem cells, stromal cells, Relevant Cells, or combinations thereof in or after any of steps (b) or (c).
 8. The method of claim 1, wherein said microvessel fragments are isolated from a tissue selected from the group consisting of skin, muscle, lung, mesentery and adipose tissue. 